Bioluminescence is a powerful, inexpensive, and non-invasive method to monitor gene expression, enzymatic activity, protein-protein interactions and response to therapeutics both in vitro and in vivo. However, bioluminescence requires the use of a luciferase derived from a small number of luminogenic organisms (e.g., fireflies), and is limited to applications and organisms where genetic manipulation is both possible and practical. In this exploratory/developmental grant, we will identify proteins in non-luminous species that are latent luciferases, capable of bioluminescent light emission when treated with an appropriate substrate. In support of this substrate-directed approach, we have identified a fruit fly protein that is revealed to be a luciferase when treated with a synthetic luciferin analog. This unprecedented result shows that the function of a protein can be fundamentally changed simply by modification of the substrate, rather than by mutation. Beyond the paradigm-shifting implications for the evolution and design of new enzymatic activities, the prospect that non-luminogenic organisms encode latent luciferases has transformative potential for the non-invasive optical imaging of endogenous processes, potentially enabling bioluminescence imaging in native cells and organisms without the need for the introduction of an exogenous luciferase.